RESUMO
Antimicrobial peptides (AMPs) are an alternative to antibiotics for treatment and prevention of infections with a lower risk of bacterial resistance. Pituitary adenylate cyclase activating polypeptide (PACAP) is an outstanding AMP with versatile effects including antimicrobial activity and modulation of immune responses. The objective of this research was to study PACAP immunomodulatory effect on rainbow trout cell lines infected with Aeromonas salmonicida. PACAP from Clarias gariepinus (PACAP1) and a modified PACAP (PACAP5) were tested. RT-qPCR results showed that il1b and il8 expression in RTgutGC was significantly downregulated while tgfb expression was upregulated after PACAP treatment. Importantly, the concentration of IL-1ß and IFN-γ increased in the conditioned media of RTS11 cells incubated with PACAP1 and exposed to A. salmonicida. There was a poor correlation between gene expression and protein concentration, suggesting a stimulation of the translation of IL-1ß protein from previously accumulated transcripts or the cleavage of accumulated IL-1ß precursor. In-silico studies of PACAP-receptor interactions showed a turn of the peptide characteristic of PACAP-PAC1 interaction, correlated with the higher number of interactions observed with this specific receptor, which is also in agreement with the higher PACAP specificity described for PAC1 compared to VPAC1 and VPACA2. Finally, the in silico analysis revealed nine amino acids related to the PACAP receptor-associated functionality.
Assuntos
Aeromonas salmonicida , Citocinas , Proteínas de Peixes , Oncorhynchus mykiss , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Animais , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Aeromonas salmonicida/fisiologia , Oncorhynchus mykiss/imunologia , Oncorhynchus mykiss/genética , Citocinas/genética , Citocinas/metabolismo , Linhagem Celular , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Peixes-Gato/imunologia , Peixes-Gato/genética , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Imunidade Inata/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genéticaRESUMO
The global aquaculture industry has significant losses each year due to disease outbreaks. Antibiotics are one of the common methods to treat fish infections, but prolonged use can lead to the emergence of resistant strains. Aeromonas spp. Infections are a common and problematic disease in fish, and members of this genera can produce antibiotic resistant strains. Antimicrobial peptides (AMPs) have emerged as an alternative method to treat and prevent infections and pituitary adenylate cyclase activating polypeptide (PACAP) is a prominent member of this family. The objective of this research was to study PACAP's direct antimicrobial activity and its toxicity in fish cells. Four synthetic variants of the natural PACAP from Clarias gariepinus were tested in addition to the natural variant. The experimental results show a different antimicrobial activity against A. salmonicida and A. hydrophila of each PACAP variant, and for the first time show dependence on the culture broth used. Furthermore, the results suggest that the underlying mechanism of PACAP antimicrobial activity includes a bacterial membrane permeabilizing effect, classifying PACAP as a membrane disruptive AMP. This study also demonstrated that the five PACAP variants evaluated showed low toxicity in vitro, at concentrations relevant for in vivo applications. Therefore, PACAP could be a promising alternative to antibiotics in the aquaculture sector.